Most myocardial infarctions are caused by coronary thrombosis. DeWood at al., N. Eng. J. Med., 303, 897 (1983). The coronary thrombi that cause myocardial infarction can be lysed by thrombolytic agents, which significantly reduce mortality. ISIS-3 Collaborative Group, Lancet, 339, 753-70 (1992); Haber, et al., Science, 243, 51-6 (1989). However, currently available thrombolytic agents may also cause haemorrhagic strokes or other bleeding. ISIS, supra: Marder, V. J. and Sherry, S., N. Engl. J. Med. 318, 1512-20 (1988); Collen, D., Am J. Cardiol., 69, 71A-81A (1992); Smitherman, T. C. Mol. Biol. Med., 8, 207-18 (1991).
Plasmin and thrombin are both enzymes that affect coronary thrombi. Plasmin is a fibrinolytic enzyme, i.e., it lyses the fibrin present in a thrombus. Currently available thrombolytic agents activate the conversion of plasminogen to the fibrinolytic enzyme plasmin. Plasmin, however, also lyses fibrinogen, resulting in a bleeding diathesis.
Thrombin, on the other hand, initiates thrombus formation by cleavage of fibrinogen and activation of platelets. Mann, K. G. and Lundbald, R. L., Hemostasis and Thrombosis: Basic Principles and Clinical Practice, (Colman, R. W., et al., eds) 2nd ed., 148-161 (1987). It is produced locally around the site of injury and is absorbed into the thrombus by interacting with fibrin. Jackson, C. M., Hemostasis and Thrombosis: Mechanism of Prothrombin Activation, (Colman, R. W., et al., eds) 2nd ed.,148-161 (1987); Fenton, J. W., II, Ann. N.Y. Acad. Sci. 322, 468-495 (1981). Fibrin-bound thrombin is enzymatically active and is slowly released from a thrombus. Liu, C. Y. et al., J. Biol. Chem. 258, 10530-5 (1979). Therefore, the inventors have identified thrombin as a transient marker for a thrombus.
Single chain urokinase-type plasminogen activator (scuPA) is a zymogen. It can be activated by plasmin cleavage between Lys 158 and IIe 159, and inactivated by thrombin cleavage between Arg 156 and Phe 157. Lijnen, H. R. et al., Semin. Throm. Hemostasis 13, 152-9 (1987); Ichinose, A. et al., J. Biol. Chem. 261, 3486-9 (1986). Thus, scuPA-initiated plasma clot lysis is apt to be regulated by plasmin or thrombin around the thrombus.
Plasmin- or thrombin-resistant scuPA mutants have been produced to study plasmin activation or thrombin inactivation of scuPA. Nelles, L., Lijnen, H. R., Collen, D., and Holmes, W. E. J. Biol. Chem., 262, 5682-9 (1987); Lijnen, R., et al. Eur. J. Biochem. 177, 575-82 (1988); Lijnen, R., et al. Eur. J. Biochem. 172, 185-8 (1988); Miyake, T.et al., J. Biochem. 104, 643-7 (1988); Eguchi, Y., et al., J Biochem. 108, 72-9 (1990). In order to increase scuPA's fibrin selectivity, chimeric plasminogen activators were constructed from an anti-fibrin antibody and low molecular weight scuPA. Both in vitro and in vivo, these activator constructs had better fibrin selectivity and higher potency than scuPA. Bode, C., et al., Science. 229, 765-7 (1985); Bode, C., et al., J. Biol. Chem. 262, 10819-23 (1987); Runge, M. S., et al., Biochemistry, 27, 1153-7 (1988). Collen, D., et al., Fibrinolysis. 3, 197-202 (1989); Dewerchin, M., et al., Eur. J. Biochem., 185, 141-9 (1989); Holvoet, P., et al., J. Biol. Chem. 266, 19717-24 (1991); Runge, M. S., et al., Proc. Natl. Acad. Sci. USA. 88, 10337-41 (1991). A chimeric immunoglobulin molecule having an antibody variable region with an antigen binding site specific for fibrin and a fibrinolytic enzyme activity region has also been prepared by recombinant DNA techniques. European Patent Application 478,366, published 1 April 1992. The preferred embodiment in that application is r-scuPA(32)-59 D8.
The foregoing research has focused on ways to increase the fibrin selectivity of potential thrombolytic agents such as the scuPA mutants and conjugates. Due to the bleeding side-effect, however, the present inventors perceive a need for thrombolytic agents that distinguish between thrombi, which lead to myocardial infarction, and hemostatic plugs, which are mainly involved in prevention of bleeding. The present inventors hypothesized that plasminogen activators such as t-PA cannot distinguish fibrin epitopes on a pathogenic thrombus from those on a hemostatic plug. According to this hypothesis, thrombolytic agents lyse both thrombi and hemostatic plugs, causing bleeding during thrombolytic therapy. See Sherry, Mod. Conc. Cardiovas. Disease. 60, 25-30 (1991).